Pseudomonas aeruginosa (MDR-PA) provides a foundation for susceptibility testing. By observing in vitro antibacterial activity and ultrastructure cthanges on MDR-PA of the effective components in the drug-containing serum of rats after the administration of Fuzheng Jiedu Huayu decoction (FJHD), we evaluated the inhibition and direct destruction effect of bacteria by TCM alone or combined with antibiotics.Drawing a growth curve of multidrug-resistant in vitro antibacterial potency of FJHD and its drug-containing serum alone or in combination with antibiotics against MDR-PA was observed. The ultrastructural changes of MDR-PA treated by FJHD combined with antibiotics were observed by transmission electron microscopy.The absorbance values of MDR-PA were determined at different detection time points, and a growth curve was drawn. After gavage with FJHD, drug-containing serum was collected from the rats. Using Imipenem/cilastatin sodium as the positive drug control, the Growth of the experimental strain manifested a lag phase in the first 1–4 h, an exponential growth phase at 5–20 h, and a plateau phase after 20 h. The best detection time during the susceptibility test was 16–20 h. The minimum inhibitory concentration (MIC) value of the FJHD extract group was 0.2 g/mL. The MIC value of the pure Imipenem/cilastatin sodium group was 16 μg/mL. The MIC values of Imipenem/cilastatin sodium + blank serum, 0.5-, 1-, and 2-fold drug-containing serum groups were all 16 μg/mL. The MIC values of Imipenem/cilastatin sodium + 4- and 8-fold drug-containing serum groups were both 8 μg/mL. By observation under a transmission electron microscope, Imipenem/cilastatin sodium + 0.5-, 1-, and 2-fold drug-containing serum groups showed bacterial structural damage. The degree of bacterial destruction was more obvious and the quantity of damaged bacteria was increased in the Imipenem/cilastatin sodium + 4- and 8-fold drug-containing serum groups.in vitro, thereby inhibiting bacterial resistance to a certain extent.Drawing the growth curve of the experimental strain had high application value for ensuring the accuracy of the drug sensitivity test results. TCM combined with antibiotics could enhance the antibacterial and direct destruction effect of bacteria  In a previous multicenter, prospective, double-blind, parallel, randomized controlled trial (Pseudomonas aeruginosa (PA). FJHD full active ingredient extract formula granules showed effectiveness in the treatment of elderly drug-resistant pneumonia in previous clinical research , which often causes drug-resistant pneumonia in the elderly, to observe the antibacterial effect of the active components in the serum of rats after gastric gavage by FJHD, and to observe the ultrastructural changes of MDR-PA after treatment with FJHD, exploring the effects of FJHD on the MDR-PA ultrastructure. The growth curve of MDR-PA was drawn, and the best detection time point was determined to ensure the accuracy of the drug sensitivity test results.Due to the extensive application of antibacterial drugs worldwide, the problem of bacterial resistance is becoming increasingly serious. The pressure to choose antimicrobial agents is increasing, and the efficacy of refractory infectious diseases caused by drug-resistant pathogens is often not satisfactory . Elderlyed trial , we founresearch . To furtFJHD full active ingredient extract formula granules were purchased from Beijing Tcmages Pharmaceutical Co., Ltd. . FJHD consists of eight herbs: scutellariae radix (Batch No: 15017541), forsythiae fructus (Batch No: 15020481), echinopsis radix (Batch No: 14019491), paeoniae radix alba (Batch No: 15012061), trichosanthis radix (Batch No: 15020191), panacis quinquefolii radix (Batch No: 15010202), patriniae radix (Batch No: 15014241), coicis semen (Batch No: 15018821), and the mixed proportion of respective herbs is illustrated in Imipenem/cilastatin sodium was purchased from the Pharmacy Department, Dongzhimen Hospital Affiliated to BUCM, and formulated into a solution with a concentration of 1,024 μg/mL using phosphate-buffered saline (PBS), as a positive drug control.Eighty male SPF grade Sprague-Dawley (SD) rats, 70–76 days old, weighing 350–460 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. .This study was conducted in accordance with the recommendations of the Guide for Care and Use of Laboratory Animals published by the U.S. National Research Council , and BeiMDR-PA clinical strain (No. 1508032) was purchased from the bacteria room of the Clinical Laboratory, Dongzhimen Hospital Affiliated to BUCM.In this study, we used the following materials: MH agar medium, MacConkey medium, and nutritional broth were purchased from Thermo Fisher Scientific Chemicals Co., Ltd. . Nutrition broth was prepared with deionized water and autoclaved.The following instruments were employed: constant temperature and humidity incubator , constant temperature oscillator , pressure steam sterilizer , low speed centrifuge , precision electronic balance , and microplate reader .The rats were divided into blank group and FJHD groups by random grouping using SPSS 19.0 at concentrations of 0.5-, 1-, 2-, 4-, and 8-fold of the equivalent dose, and formulated into a Chinese medicine suspension with concentrations of 0.5, 1, 2, 4, and 8 g/mL. There were 10 experimental rats in each group. The blank group was given normal saline. The FJHD groups were given FJHD full active ingredient extract formula granules. Each group was administered twice a day for 5 days.At 1 h after the last gavage, SD rats were anesthetized with chloral hydrate. With a sterilized technique, blood was collected through their abdominal aorta and centrifuged at 1,610 × g for 10 min to separate the drug-containing serum. The same group of serum was mixed with each other. Then the serum was inactivated at 56°C for 30 min in water bath, filtered through a 0.22-μm pore size filter, dispensed into a cryotube, and stored in a –70°C refrigerator. Mixing, filtration, and dispensing were all performed in a biosafety cabinet.7 cfu/mL by a turbidimetric method with sterile physiological saline.A small amount of MDR-PA cells  cryopreserved in an –80°C refrigerator were inoculated on MacConkey agar medium and cultured at 35°C for 18–24 h to activate the strain. The next day, the MDR-PA bacterial solution was adjusted to a concentration of 1 × 105 cfu/mL) was added. Tubes 1–3 were parallel test tubes, and the 4th tube was a blank control tube with no bacterial fluid, as a negative control. The tubes were placed in a constant temperature oscillator for shaking culture . In accordance with relevant literature  values of different doses of Chinese herbal extracts and drug-containing serum on MDR-PA were determined by tube dilution method  . All of th tube served as the negative control and did not have bacterial liquid. The 9th tube served as the model control and did not have Chinese herbal extract (or drug-containing serum). The prepared 0.5 Mc concentration solution was diluted by 10-fold, corresponding to a bacterial liquid concentration of 1.5 × 107 cfu/mL, followed by the addition of 0.1 mL to each tube. After shaking for 30 s, the MDR-PA cells were cultured in a 35°C incubator, and growth of the bacteria was observed after 24 h. The composition of the test tube system is shown in Nine sterile tubes were taken from each group; 1 mL broth was added to tubes 1–7 and 2 mL was added to tubes 8–9. Volumes of 1, 0.8, 0.6, 0.4, 0.3, 0.2, and 0.1 mL Chinese herbal extract/drug-containing serum were added to tubes 1–7, and broth was added to a total volume of 2 mL in tubes 2–7. The 8st tube . After the 1st tube was mixed, 1 mL was taken out and added to the 2nd tube, and after mixing in the 2nd tube, 1 mL was taken out and added to the 3rd tube, and the operation was repeated until the 7th tube. After the 7th tube was mixed, 1 mL was taken out and discarded. All of the pipetting operations were performed normatively in a biosafety cabinet using a 1,000 μL pipette specification.Nine sterile tubes were taken from each group; 1 mL broth was added to sterile tubes 1–7, 2 mL was added to tubes 8 and 9, and 1 mL Imipenem/cilastatin sodium solution was added to the 1Imipenem/cilastatin sodium group: 1 mL broth was added to each of the 1–7 tubes.Imipenem/cilastatin sodium + drug-containing serum group: 0.4 mL of the drug-containing serum was added to each of the 1–7 tubes, and then the broth was added to make up the 2 mL system in the tubes.7 cfu/mL, and 0.1 mL was added to tubes 1–7 and 9. The 8th tube only had broth as a negative control, and the 9th tube had broth and bacterial solution as a model control. The solution in every tube was mixed well and placed in a 35°C incubator. The growth of bacteria was observed after 24 h. The test tube system of the Imipenem/cilastatin sodium + drug-containing serum groups is shown in The prepared 0.5 Mc concentration solution was diluted 10-fold, corresponding to a bacterial concentration of 1.5 x 10Each group of tubes in the tube dilution method was cultured in a constant temperature and humidity incubator at 35°C for 24 h. With the test tube of Imipenem/cilastatin sodium MIC set as a standard, 3 mL PBS was added to the next test tube of each group, followed by centrifugation at 3,000 rpm for 10 min, after which the supernatant was discarded. The process was repeated three times to remove the serum, broth, antibiotics. Then the precipitate was fixed in glutaraldehyde, embedded, sectioned, and stained. The ultrastructure of the MDR-PA was observed by transmission electron microscopy and the nature and extent of the ultrastructural lesions were recorded.The OD570nm values of the test tubes and negative control tubes of MDR-PA cultured for 24 h were measured. The results are shown in nm value. The higher the OD570nm value, the higher the concentration of bacterial solution, indicating that the bacteria grew more. As seen in nm value gradually increased, indicating that the MDR-PA in nutrient broth was growing and multiplying, and the amount of bacteria was gradually increasing. A negative control tube was set up in the test. The OD570nm value in the table = OD570nm value of the MDR-PA test tube - the OD570nm value of the negative control tube, and the effect of the nutrient broth itself on the OD570nm value was excluded.The concentration of the bacterial solution was reflected by the OD 570nm value as the ordinate, the relationship between the concentration and the OD570nm value of the MDR-PA at different time points is shown in The culture time was taken as the abscissa and the OD570As shown in extract group: 1st tube 1 g/mL, 2nd tube 0.8 g/mL, 3rd tube 0.6 g/mL, 4th tube 0.4 g/mL, 5th tube 0.3 g/mL, 6th tube 0.2 g/mL, 7th tube 0.1 g/mL. Tubes 1–6 of the Chinese herbal extract group were clear and light yellow, and the 7th tube was turbid, with bacterial growth. The model control tube was obviously turbid. The negative control tube was clear. The MIC of the Chinese medicine extract group was 0.2 g/mL. The test tube reaction system results of the Chinese medicine extract group are shown in FJHD Chinese medicine th tubes all showed varying degrees of turbidity and bacterial growth. The model control tube was obviously turbid. The negative control tube was clear. All of the simple drug-containing serum groups showed no obvious antibacterial effect. The test tube reaction system results of each drug-containing serum group are shown in Simple drug-containing serum groups: The 1–7th tube appeared turbid. The 7th tube was turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of the simple Imipenem/cilastatin sodium group was 16 μg/mL.The simple Imipenem/cilastatin sodium group included tubes 1–5, which were clear and light yellow. The 6th tube appeared turbid. The 7th tube was turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of the Imipenem/cilastatin sodium + blank serum group was 16 μg/mL.In the Imipenem/cilastatin sodium + blank serum group, tubes 1–5 were clear and light yellow. The 6th tube appeared turbid. The 7th tube was turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of Imipenem/cilastatin sodium + 0.5-fold drug-containing serum group was 16 μg/mL.In the Imipenem/cilastatin sodium + 0.5-fold drug-containing serum group, tubes 1–5 were clear and light yellow. The 6th tube appeared turbid. The 7th tube was turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of Imipenem/cilastatin sodium + 1-fold drug-containing serum group was 16 μg/mL.In the Imipenem/cilastatin sodium + 1-fold drug-containing serum group, tubes 1–5 were clear and light yellow. The 6th tube appeared turbid. The 7th tube was turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of the Imipenem/cilastatin sodium + 2-fold drug-containing serum group was 16 μg/mL.In the Imipenem/cilastatin sodium + 2-fold drug-containing serum group, tubes 1–5 were clear and light yellow. The 6th tube appeared turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of Imipenem/cilastatin sodium + 4-fold drug-containing serum group was 8 μg/mL.In the Imipenem/cilastatin sodium + 4-fold drug-containing serum group, tubes 1–6 were clear and light yellow. The 7th tube appeared turbid. The model control tube was obviously turbid. The negative control tube was clear. The MIC of Imipenem/cilastatin sodium + 8-fold drug-containing serum group was 8 μg/mL. The test tube reaction system results of each Imipenem/cilastatin sodium+ drug-containing serum group are shown in In the Imipenem/cilastatin sodium + 8-fold drug-containing serum group, tubes 1–6 were clear and light yellow. The 7th tube. According to the results of the test tube dilution method, observation specimens were prepared with test tube reaction system after the 6th tube of each group. The model control group was obtained by culturing MDR-PA in pure broth, using the tube dilution method.In the test tube dilution method, the MIC value of Imipenem/cilastatin sodium was 16 μg/mL in the 5In the model control group, MDR-PA cells growing in pure broth showed characteristic shapes of Gram-negative bacteria, with uniform electron density and a clear extracellular outer membrane and intracellular protoplast.In the simple Imipenem/cilastatin sodium group, the outer membrane was damaged and blurred in the polar region of MDR-PA cells. The inner and outer membrane integrity was damaged. Aggregated dense particles were observed in the protoplast. The cellular inclusions disappeared but outer membranes remained in some cells, exhibiting an empty shell state.In the Imipenem/cilastatin sodium + 0.5-fold drug-containing serum group, the outer membrane was damaged in the polar region of MDR-PA cells, and part of the outer membrane ruptured. Aggregated dense particles were observed in the protoplast. The cellular inclusions disappeared but the outer membranes remained in some cells. The cell surfaces showed vacuoles. The inner and outer membrane integrity was damaged, and the two membrane structures contained cytoplasm, which protruded from the surface of the bacteria.In the Imipenem/cilastatin sodium + 1-fold drug-containing serum group, the inner and outer membrane were damaged.In the Imipenem/cilastatin sodium + 2-fold drug-containing serum group, the outer membrane was damaged and partially ruptured. Aggregated dense particles and vacuoles were observed in the protoplast. Cellular inclusions totally disappeared in some cells. Cell surfaces showed vesicles that appeared as rough structural spots that could have been cellular constituents leaking from cells.In the Imipenem/cilastatin sodium + 4-fold drug-containing serum group, the outer membrane was damaged in the polar region and partially ruptured. Aggregated dense particles were observed in the protoplast. Cellular inclusions totally disappeared in some cells. The inner and outer membrane integrity was damaged.In the Imipenem/cilastatin sodium + 8-fold drug-containing serum group, the outer membrane was damaged in the polar region and partially ruptured. Aggregated dense particles were observed in the protoplast. The inner and outer membrane integrity was damaged. Cell surfaces showed vesicles. The cells were severely damaged. The Morphological changes of MDR-PA in all of the groups are shown in According to the photomicrograph of the electron micrograph, Imipenem/cilastatin sodium + 0.5-fold, 1-fold, and 2-fold drug-containing serum groups showed bacterial structure damage, similar to the simple Imipenem/cilastatin sodium group, and there was no significant difference. The degree of bacterial destruction was more obvious in the Imipenem/cilastatin sodium + 4-fold and 8-fold drug-containing serum groups than in the simple Imipenem/cilastatin sodium group, with an increasing trend. The region where the bacteria distribution density was relatively uniform under 30,000 times of field of view was selected and locked. In the region the total number of visible bacteria, the number of damaged bacteria and the bacterial damage ratio was counted. The statistical results of the model control group  was greater than Imipenem/cilastatin sodium + 4-fold serum and greater than Imipenem/cilastatin sodium + 2-fold serum and greater than the Imipenem/cilastatin sodium group. A suitable system for drug-containing serum was 20%. In the in vitro experimental study of TCM serum, the amount of serum added was an important factor affecting the experimental results. Therefore, it was of great significance to explore the optimal reaction system in the experiment. In the preliminary experiment, the research group tried 40% (0.8 mL), 30% (0.6 mL), and 20% (0.4 mL) reaction system. As a result, in the 20% reaction system, the antibacterial effect of Imipenem/cilastatin sodium combined with drug-containing serum was the best. According to the relevant literature, there was no clear regulation on the concentration of drug-containing serum about in vitro experiments. Most of the drug-containing serum accounted for 5% to 20% of the reaction system , as the fold of equivalent dose drug-containing serum increased, the peripheral secretory granules of bacteria increased. Under transmission electron microscopy, the model control group and simple Imipenem/cilastatin sodium group showed no secretory granules at bacterial periphery. Imipenem/cilastatin sodium + 0.5-fold drug-containing serum group showed individual granules, and Imipenem/cilastatin sodium + 2-fold drug-containing serum group showed a few particles. Imipenem/cilastatin sodium + 8-fold drug-containing serum group showed more exocrine particles in the periphery of the bacteria. The production of exocrine granules of Gram-negative bacteria was considered to be a way for live bacteria to release endotoxin . The endTherefore, this experiment demonstrated that, when the drug-containing serum combined with antibiotics acted on MDR-PA, in addition to directly destroying the bacterial structure, causing damage and lysis, the active ingredients in the drug-containing serum could also release endotoxin from the bacteria to stimulate lymphocytes and produce an immune response that could clear bacteria.This experiment initially showed that the antibacterial effect of TCM combined with antibiotics was stronger than simple antibiotics against multidrug-resistant pathogens. However, the interventional effect of TCM on drug-resistant mechanism of pathogens has not yet been deeply explored. The intervention mechanism of TCM against bacterial resistance should be further studied, and the active ingredients of TCM should be further revealed to provide a more reliable experimental basis for the prevention and treatment of respiratory drug-resistant infectious diseases.The datasets generated for this study are available on request to the corresponding authors.The animal study was reviewed and approved by the Beijing University of Chinese Medicine Medical and Experimental Animal Ethics Committee.HX and CW provided detailed guidance on the implementation of this experiment. HX wrote, revised, and improved the paper. CL and MW participated in the experiment and edited the manuscript. GL, JM, LL, MeC, SZ, and YW performed the experiments. ML, HW, MiC, XY, and YL participated in part of the experiments.This work was funded by the Research Fund for the Doctoral Program of Higher Education of China (No. 20130013120004), the National Natural Science Foundation of China (Nos. 81473661 and 81573924), and Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine Qingmiao Talent Program (No. DZMYS-201608).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pyropia yezoensis, commonly known as “Nori” or “Laver” is an economically important marine crop. In natural or selected populations of P. yezoensis, coloration mutants are frequently observed. Various coloration mutants are excellent materials for genetic research and study photosynthesis. However, the candidate gene controlling the Pyropia coloration phenotype remains unclear to date. QTL-seq, in combination with kompetitive allele-specific PCR (KASP) and RNA-seq, can be generally applied to population genomics studies to rapidly identify genes that are responsible for phenotypes showing extremely opposite traits. Through cross experiments between the wild line RZ and red-mutant HT, offsprings with 1–4 sectors chimeric blade were generated. Statistical analyses revealed that the red thallus coloration phenotype is conferred by a single nuclear allele. Two-pair populations, consisting of 24 and 56 wild-type/red-type single-genotype sectors from F1 progeny, were used in QTL-seq to detect a genomic region in P. yezoensis harboring the red coloration locus. Based on a high-quality genome, we first identified the candidate region within a 3.30-Mb region at the end of chromosome 1. Linkage map-based QTL analysis was used to confirm the candidate region identified by QTL-seq. Then, four KASP markers developed in this region were used to narrow down the candidate region to a 1.42-Mb region. Finally, we conducted RNA-seq to focus on 13 differentially expressed genes and further predicted rcl-1, which contains one non-synonymous SNP [A/C] in the coding region that could be regulating red thallus coloration in P. yezoensis. Our results provide novel insights into the underlying mechanism controlling blade coloration, which is a desirable trait in algae. Blade color mutations endow plants with various colors that can influence physiological changes in plants and increase the economic value of agricultural products and ornamental plants . Blade cArabidopsis thaliana (Chl) and carotenoids  (–2⋅s–1 at 8 ± 1°C and a 12:12 light:dark (L:D) photoperiod. Free-living conchocelis filamentous (sporophytes) were cultured in bubbling natural seawater with PES under 20 μmol photons m–2⋅s–1 at 20 ± 1°C and a 12:12 light:dark (L:D) photoperiod. HT as paternal was crossed to RZ as maternal by the methods by 2) tests for goodness of fit   and the 440 (RZ) , were uss of fit .For each sector of four-sectored chimeras and the polar sector of three-sectored chimeras, all cells of each sector have the same genotype. Therefore, we directly used single-genotype sectors for the subsequent genetic analysis.a (Chla) were subsequently measured. For Chla, about 0.1 g of each sample was ground in 5 mL of 95% ethanol and extracted at 4°C in darkness for 24 h. For PE and PC, 0.1 g of each sample was ground in 10 mL of extraction buffer  at 4°C. The extract was centrifuged at 5,000 g for 10 min and then used to determine the contents of Chla, PE, PC, and APC using an ultraviolet spectrophotometer . Chla content was analyzed according to For pigment content analysis between RZ and HT, blades from two parents with the same living conditions were sampled, samples were frozen at −80°C before pigment extraction, and the contents of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), and chlorophyll Total DNA from single-genotype color sectors or parents’ blades was extracted using a Plant Genomic DNA Kit  following the manufacturer’s protocol. DNA concentration and quality were examined with the Qubit.2.0 fluorometer (Invitrogen) and 1% agarose gel electrophoresis and a N60 UV-Vis spectrophotometer . DNA was subsequently stored at −80°C until further analysis.The F1 single-genotype color sectors mentioned earlier were used in QTL-seq. A high-quality reference genome consisting of three chromosomes was from RZ line blades’ material . A total of five DNA pools were further constructed, namely, P1, RT24, WT24, RT56, and WT56. The P1 pool was from the blades of HT ; two pairs of mixed pools RT24-WT24 and RT56-WT56 were constructed by equally mixing 24 or 56 F1 single-genotype sectors with different color traits (WT or RT). Paired-end sequencing libraries with insert sizes of approximately 350 bp and a read length of 150 bp were subjected to whole-genome resequencing with Illumina HiSeq 2000 platform .P. yezoensis genome sequence using the Burrows-Wheeler alignment tool (BWA)  and GATK software were used to detect single-nucleotide polymorphisms (SNPs) and InDels  . Picard d InDels . ANNOVERd InDels . SNPs/InHomozygous SNPs/InDels between two parents were extracted from the VCF files. Using the RZ genome as reference, we calculated the statistic read number for this reference parent in the offspring pool. Then, SNP/InDel-index and the Δ(SNP/InDel-index) values were calculated to identify candidate genomic regions associated with the red-type trait of HT . The ΔS. We calc2. The boundary of each QTL was determined by 1.5-LOD drop support intervals. The regional genes were annotated and analyzed on the base of previous analysis of P. yezoensis in our lab.In order to confirm the accuracy of candidate region identified by QTL-seq, another 84 single-genotype color sectors from the F1 offspring were used to conduct linkage map-based QTL analysis. Markers for the construction of the genetic map were screened using the same protocol earlier described and stricter filter criteria  ≥ 0.05). QTL mapping was analyzed using R/qtl software . IntervaUsing the information from the QTL-seq analysis of SNPs related to the candidate region, selected SNPs were confirmed using the Kompetitive Allele Specific PCR (KASP) method following the manufacturer’s instructions . A totalFor gene expression analysis of the candidate region, blades from two parents living in the same condition were sampled for RNA-seq. Each parent sampling was conducted using three biological replicates. Total RNA was extracted using a Plant RNA Kit (OMEGA), and the first-strand cDNA was prepared using a HiScript II Q RT SuperMix for the qPCR kit (Vazyme Biotech). RNA quality was assessed using an RNA Nano 6000 Assay Kit and Bioanalyzer 2100 system (Agilent Technologies). Sequencing libraries were generated using a VAHTS Total RNA-Seq Library Preparation Kit (Vazyme Biotech). Then, the libraries were paired-end sequenced on an Illumina HiSeq 2000 platform  with the read length of 150 bp.The quality of raw sequencing reads was evaluated using FastQC  and TrimP. yezoensis sequence and gene functional annotations were deposited to DDBJ/ENA/GenBank as accession WMLA00000000. Conserved domains were identified in NCBI3 and Pfam4 databases. Amino acid sequences were aligned using MEGA 7  gene was used as reference gene. The sequences of the primers used are listed in –ΔΔCt method was used to calculate relative gene expression values.RNA sampling and cDNA synthesis were as earlier described. Three biological and technical replicates were used for qRT-PCR analysis. Quantitative RT-PCR (qRT-PCR) was conducted using a ChamQ SYBR color qPCR Master Mix (Vazyme Biotech). The primers for qRT-PCR were designed using Primer 5.0 software. And the selected genes were verified using the Light-Cycle5.Phylogenetic neighbor-joining (NJ) trees were constructed using MEGA 7 software using the bootstrap method and 1,000 replications. The phylogenetic relationships of the studied red algae were assessed using TimeTree v3.0After crossing RZ and HT , carposp2 = 1.90 < χ0.052 = 3.84; The color phenotypes and blade types of F1 gametophytic blades from the heterozygous conchocelis in the cross between RZ and HT were counted . Eight tChla than HT (P < 0.05). PE and APC concentrations in HT were, respectively, 34.53% (P < 0.01) and 35.41% (P < 0.05) higher than RZ. However, PC concentrations in HT were 10.29% higher than RZ, which were not significantly different (P > 0.05). The ratios of pigments in blades of the RZ and the HT were also compared . However, no significant differences in the APC/PC and PE/APC between HT and RZ were observed (P > 0.05).Pigment contents varied between the two parents . Remarkably, only one non-synonymous SNP [A/C] in the coding region of Py08429 was located in the final candidate region.A total of 36 SNP sites at physical position 39.70–43.00 Mb of chromosome 1 were identified by QTL-seq. To further narrow down the region, we developed 10 KASP markers, and finally, four valid KASP markers in this region were used for genotyping an additional set with 328 samples . Recombi2(fold_change)| >1), including 605 annotated genes and 854 unannotated genes (P < 0.05) were in agreement with the RNA-seq differentially expressed analysis except for Py04880, Py08094, and Py06313  and directly associated with pigments, was upregulated in HT compared to RZ; this finding coincides with the pigment content results; while in the chlorophyll metabolic pathway, the expression level of magnesium chelatase subunit H (Py02527) and geranylgeranyl reductase  decreased in HT compared to RZ; although these three genes were not included in the candidate region described above.Among these genes, one gene, which is predicted to be the gamma subunit of phycoerythrin (2(fold_change)| >1) (Py08429 gene exhibiting pronounced upregulation (>6-folds) in red mutant (HT) | >1) . Combineant (HT) , 5 was cPy08429 from both RZ and HT. The results suggested that the presence of one exon and the predicted coding sequence (CDS) of the cloned Py08429 cDNA was 1,338 bp, with a predicted corresponding protein length of 446 amino acids. Alignment of the Py08429 sequence between the RZ and HT revealed a single non-synonymous A→C mutant in the exon, resulting in the conversion of a glutamine  to proline  at residue 331. Searching in the conserved domain database (CDD) and BLAST in NCBI did not reveal any annotation information on Py08429.We cloned P. yezoensis lines were selected, and these Py08429 sequences were determined. All these WT P. yezoensis lines shared the RZ protein sequence in this site  showed rcl-1 homologs.To better predict the information of  species  using BLPha_Ph00145, 84.73% sequence identity with Pum_OSX75132.1, 42.21% sequence identity with Ppu_contig_2019.17, 42.54 and 37.89% sequence identity with Gsu_XP_005702418.1 and Gsu_XP_005708968.1, respectively, and 27.88% sequence identity with Cme_XP_005539389.1. The site of rcl-1 protein where the non-synonymous mutation occurred is highly conserved . The life history and generation alteration in Pyropia are quite different from those of higher plants  released from filaments propagated from carpospores. During the germination of the conchospores, meiosis occurs and the haploid, ordered tetrads are generated  for pooling. As for other qualitative and quantitative traits that need to be determined by measuring intact individuals such as length and width, it is still necessary to induce monospores and culture these into intact leaves for further genetic analysis. The advantages of QTL-seq vis-a-vis other available traditional QTL mapping approaches to identify major QTLs governing fruit and leaf color in higher plants  have recently been reported , evaluating traits, and then using adjusted QTL-seq, and combined KASP and RNA-seq methods for trait location. Members of Bangiales have similar life cycle and characteristics, and this study has demonstrated the effectiveness of this strategy and provided ideas for other traits controlled by single nuclear-encoded genes or some threshold traits in Bangiales and even algae.SiYGL1 gene encoding a magnesium-chelatase D subunit (CHLD), OsYGL1 gene encoding the chlorophyll (Chl) synthase, and ZmYGL1 predicted to encode a cpSRP43 protein are associated with chlorophyll biosynthesis, and mutations in these genes result in Chl-defective yellow-green leaf mutants  is essential to the synthesis of carotenoid precursors of abscisic acid (ABA), and transgenic rice plants harboring the PDS-RNAi construct exhibit an albino phenotype  and AtCGA1 (cytokinin-responsive gata1)  . In the ed algae .Pyroipa usually contains three major pigments, namely, phycoerythrin, phycocyanin, and chlorophyll a , the pink mutant with high PE/Chla and PE/PC ratios. Here, we found that the content of Chla in HT was significantly lower than RZ blades, and among the pigment ratios, the PE/Chla, PC/Chla and APC/Chla of HT was extremely significantly higher than RZ. These results reveal that the observed color change is mainly caused by a decrease in Chla content. RNA-seq also show that the expression level of magnesium chelatase subunit H and the geranylgeranyl reductase (ChlP) gene significantly decreased in HT compared to RZ, which could directly decrease the level of Chla content in RZ. In addition, we found that the content of PE in HT was significantly upregulated compared to RZ, and the expression level of phycoerythrin subunit in HT was higher than RZ, which coincided with the increase in pigment content. Some investigations have found that the decrease in the contents of chlorophyll and total carotenoids can be accompanied by an increase in phycoerythrin abundance of phycobilisomes (rcl-1 may be related to the Chla metabolic pathways, which results in a decrease in Chla content and in turn increasing PE content, thereby leading to color changes. However, it is unclear if this qualitative gene that controls the coloration of the red-mutant blades is derived from the structural gene or transcription factor gene of the pigment biosynthetic pathway. Therefore, it is not possible to exclude the presence of rcl-1 that acts as a switch in the coloration of red-mutant blades upstream of pigment metabolic pathway-related genes or transcription factors.Unlike pigment components in higher plants, the gametophytic blade of ophyll a . Blade cophyll a . Yan et ilisomes . Some geilisomes . This phrcl-1’s homologs among 18 published genome databases from a range of plant species, we found that only red algae contain rcl-1 homologs. The high degree of protein sequence conservation with homologs from red algae species further suggests the importance of the rcl-1 non-synonymous mutations. The gene associated with red coloration in P. yezoensis and its absence in higher plants and green algae also suggests that rcl-1 may also be related to the unique pigment composition system and even the photosystem of red algae. The results of previous investigations and the present study clearly indicate the need for more detailed functional validation and detailed molecular characterization of this gene to understand its definite role in color regulation in P. yezoensis. Gene editing technology in Pyropia is currently in progress, and in the future, this technology may help us fully confirm and understand its function. Therefore, a red coloration rcl-1 gene could be potentially utilized as a molecular marker for genetic analysis and marker-assisted genetic improvement of Pyropia.By searching for P. yezoensis. First, genetic analysis showed that this trait followed Mendel’s laws, indicating that it is a qualitative trait. Then, we used a combination of QTL-seq and KASP to narrow down the region containing the rcl-1 gene to a segment of 1.42 Mb in size. Combined with RNA-seq, rcl-1, which contains one non-synonymous SNP [A/C] in the coding region and different expression levels between RZ and HT, has been shown to be essential for regulating red coloration in P. yezoensis. This is the first report on fine mapping functional genes in red algae and the elucidation of the relationship between rcl-1 genetic locus and pigment regulation.In this study, we identified the genetic locus that regulates the red coloration in SRR11820821–SRR11820826.The RNA-seq data for this study can be found in the National Center for Biotechnology Information Sequence Read Archive under the accession numbers XY and YM designed the study. XY, LW, and BS conducted the experiments. XY and KX analyzed the data. XY wrote the manuscript, which was revised by YM, FK, DW, and XT. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Edwardsiella piscicida  is the leading pathogen threatening worldwide fresh and seawater aquaculture industries and has been considered as a model organism for studying intracellular and systemic infections. However, the role of type II TA systems are completely unknown in aquatic pathogenic bacteria. In this study, we identified and characterized a type II TA system, YefM-YoeB, of E. piscicida, where YefM is the antitoxin and YoeB is the toxin. yefM and yoeB are co-expressed in a bicistronic operon. When expressed in E. coli, YoeB cause bacterial growth arrest, which was restored by the addition of YefM. To investigate the biological role of the TA system, two markerless yoeB and yefM-yoeB in-frame mutant strains, TX01ΔyoeB and TX01ΔyefM-yoeB, were constructed, respectively. Compared to the wild strain TX01, TX01ΔyefM-yoeB exhibited markedly reduced resistance against oxidative stress and antibiotic, and markedly reduced ability to form persistent bacteria. The deletion of yefM-yoeB enhanced the bacterial ability of high temperature tolerance, biofilm formation, and host serum resistance, which is the first study about the relationship between type II TA system and serum resistance. In vitro infection experiment showed that the inactivation of yefM-yoeB greatly enhanced bacterial capability of adhesion in host cells. Consistently, in vivo experiment suggested that the yefM-yoeB mutation had an obvious positive effect on bacteria dissemination of fish tissues and general virulence. Introduction of a trans-expressed yefM-yoeB restored the virulence of TX01ΔyefM-yoeB. These findings suggest that YefM-YoeB is involved in responding adverse circumstance and pathogenicity of E. piscicida. In addition, we found that YefM-YoeB negatively autoregulated the expression of yefM-yoeB and YefM could directly bind with own promoter. This study provides first insights into the biological activity of type II TA system YefM-YoeB in aquatic pathogenic bacteria and contributes to understand the pathogenesis of E. piscicida.The emergence of drug resistant bacteria is a tricky and confronted problem in modern medicine, and one of important reasons is the widespread of toxin-antitoxin (TA) systems in pathogenic bacteria.  Edwardsiella was isolated from infected humans and animals and identified as a new genus of Enterobacteriaceae in 1965  is recognized as one of the most severe pathogens in cultivating fishery  and heterohexamer (YoeB-YefM2-YefM2-YoeB)  . In S. pormation . In E. cl stress  and YefMl stress . CurrentE. coli BL21 (DE3) was purchased from TransGen . E. coli S17-1λpir was purchased from Biomedal . E. piscicida TX01 was isolated from diseased fish. Bacteria were cultured in Luria-Bertani broth (LB) at 37°C (for E. coli) or 28°C (for E. piscicida). Where indicated, Ampicillin, Kanamycin, chloramphenicol, tetracycline, and polymyxin B, were supplemented at the concentration of 100, 50, 30, 15, and 100 μg/mL  and HP Total RNA kit . Total RNA was used to synthesize the cDNA with random primers and PrimeScript Reverse Transcriptase . cDNA was used as template for PCR amplification, simultaneously, genomic DNA as a positive control and total RNA as a negative control for the PCR. The PCR were analyzed with the primers YoeBRTF/YoeBRTR for yoeB (control) and YefM-YoeBRTF/YefM-YoeBRTR for yefM-yoeB. Primers YefM-YoeBRTF/YefM-YoeBRTR were annealed to the 5′ end of yefM and 3′ end of YoeB coding region, respectively, and Gel electrophoresis was analyzed the PCR products amplified with genomic DNA, cDNA, and RNA. The primers used in this study were listed in Overnight cultures of yefM and yoeB were amplified with the primer pairs YefMF4/YefMR4 and YoeBF4/YoeBR4, respectively. The amplified fragments were inserted into the pBT3 (E. coli BL21 (DE3), which resulted in BL21/pETYoeB, BL21/pTYefM+pETYoeB, and BL21/pTYefM+pET. These strains were cultured in LB with or without 0.5 mM IPTG and bacterial growth curve was monitored for 12 h  of yoeB was performed by overlap extension PCR as follows: the first overlap PCR was performed with the primer pair YoeBKOF1/R1, the second overlap PCR was performed with the primer pair YoeBKOF2/R2, and the fusion PCR was performed with the primer pair YoeBKOF1/R2. The PCR products amplified by primer pair YoeBKOF1/R2 were inserted into the suicide plasmid pDM4 at the BglII site, resulting in pDMYoeB. S17-1λpir was transformed with pDMYoeB and the transformants were conjugated with TX01 as described previously  of yefM-yoeB was performed by overlap PCR. The experiment was performed as described above. To constructed the complementary strain TX01ΔyefM-yoeBC, yefM-yoeB was amplified by PCR with primers YefM-YoeBC-F/R, was inserted into the plasmid pJR21 at the PmeI site, resulting in pJRYefM-YoeB. Then pJRYefM-YoeB was transformed into TX01ΔyefM-yoeB by conjugation. The complementary strain was named TX01ΔyefM-yoeBC.The primers used in this study were listed in eviously . The trayoeB, TX01ΔyefM-yoeB, and TX01ΔyefM-yoeBC were cultured in LB medium to exponential phase, then bacteria were transferred to fresh LB medium and cultured at 28 and 40°C. Bacterial cell density was measured at different time points by determining absorbance at OD600 as previously described . After incubating at 28°C for 60 min, the mixtures were serially diluted and plated in triplicate on LB agar plates. The plates were incubated at 28°C for 24 h, and the colonies that appeared on the plates were enumerated. The survival rate was calculated as follows: [(number of H2O2–treated cells)/(number of control cells)] × 100%  were incubated with 50 μL non-immune fish serum or PBS (control) for 60 min. The mixtures were serially diluted and plated in triplicate on LB agar plates. The plates were incubated at 28°C for 24 h, and the colonies that appeared on the plates were enumerated. The survival rate was calculated as follows: [(number of serum-treated cells)/(number of control cells)] × 100%  for observation of biofilms. The staining procedure involved incubation for 20 min at room temperature in the dark. The biofilms were observed using a Leica TCS-SP2-AOBS-UV confocal laser scanning microscope equipped with an argon ion laser. The experimental procedures were performed three times  was performed as described by ee times .yoeB, and TX01ΔyefM-yoeB were cultivated to OD600 = 0.5 (log phase), prepared a bacterial suspension (108 CFU/mL) and plated it evenly on the MH solid plates. Thirty kinds of drug sensitive tablets  were attached to the plates, and three types of them were placed on each plate. After that, the plates were cultivated at 28°C for 24 h. Finally, the diameters of the inhibition zone on the plates were measured. The experiment was performed three times.The antibiotic drug susceptibility test was carried out in accordance with the operating standards of the paper diffusion method  recommended by the Clinical Laboratory Standards Institute (CLSI). TX01, TX01ΔyefM-yoeB has an effect on the persister formation, TX01, TX01ΔyoeB, and TX01ΔyefM-yoeB were cultured overnight and the culture solution was diluted with 1:100 in LB. Bacterial culture was incubated at 28°C on a shaker at 200 rpm until the absorbance at 600 nm reached 0.5 (logarithmic phase). Then, the bacteria were diluted serially with LB. The bacteria  were exposed to a lethal dose of 50 × MIC of chloramphenicol  with Japanese flounder gill cells (FG cells) was performed as below. FG cells were cultured in 96-well cell culture plates to a monolayer and mixed with 100 μL strain (1 × 106 CFU/mL) at a multiplicity of infection (MOI) of 10:1. After incubation at 25°C for 1 and 2 h, the plates were washed three times with PBS. To determine the number of bacterial cells associated with the entire FG cell, the washed FG cells were lysed with 200 μL of 1% (vol/vol) Triton X-100 in PBS, and the number of bacteria was counted by dilution plating  was performed as previously (E. piscicida (1 × 106 CFU) was added into RAW264.7 cells cultured in Dulbecco’s minimal Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS)  at a MOI of 10:1. After incubating at 28°C for 2 h, the plates were washed with PBS and extracellular E. piscicida was killed by adding gentamicin (100 μg/mL) for 2 h. Then, the cells were washed with PBS and cultured in fresh DMEM containing 10 μg/mL gentamicin for 0, 2, 4, and 6 h. For detecting the number of survival bacteria in RAW264.7 cells at different time point, cells were lysed and lysate were plated on LB agar plates as described above.Bacterial replication in murine monocyte-macrophage cells  were infected by intramuscularly (i.m.) with 50 μL TX01, TX01ΔyoeB, TX01ΔyefM-yoeB, TX01ΔyefM-yoeBC, and PBS, respectively. At 24 and 48 h post-infection (hpi), spleen from three fish were taken aseptically. The bacterial recovery from the spleen was determined as reported previously  were purchased from a commercial fish farm of Hainan. The fish were maintained at 25 ± 1°C in aerated water and fed daily with commercial dry pellets. Fish were acclimatized in the laboratory for 2 weeks before experimental manipulation . To makeeviously . The resyefM-yoeB (the 345 bp of upstream of the yefM-yoeB operon), P345, was cloned with the primer pair YefM-YoeBP-F/YefM-YoeBP-R and inserted into the SwaI site of pSC11, a promoter probe plasmid   were transformed into DH5α/pSCP345, respectively, which resulted in DH5α/pSCP345/pTYefM, DH5α/pSCP345/pTYefM-YoeB, and DH5α/pSCP345/pT. The transformants were cultured on X-gal plates and used for β-galactosidase assay as reported previously  . To conseviously .yefM were amplified with the primer pair YefMPro-F/YefMPro-R and then the PCR segments were inserted into the protein expression plasmid pET32a, resulting in pET32aYefM. The expressions and purifications of rYefM protein and control protein rTrx were performed as previously  and the negative control was a mixture of rTrx and nucleic acid probe. The samples were then separated by electrophoresis in non-denaturing 8% polyacrylamide gels (An electrophoresis mobility shift assay (EMSA) was performed as below. The DNA fragment of the speculative promoter was amplified by PCR and labeled with carboxyfluorescein . The labeled DNA was mixed with rYefM and incubated at 37°C for 30 min in 20 μL of binding buffer . Statistical significance was defined as P < 0.05 . Data were analyzed with analysis of variance (ANOVA). Data are expressed as the mean ± standard error of the mean (SEM). Error bars indicate the Standard Error of Mean (SEM)  was conducted using total RNA, complementary DNA (cDNA), and genomic DNA (gDNA) of E. piscicida as templates. The results showed that the predicted PCR products of 447 bp were observed in cDNA and gDNA templates, but not in RNA template  of chloramphenicol was used to treat these strains. Viable bacteria were determined and the result showed the number of persistence bacteria of TX01ΔyoeB and TX01ΔyefM-yoeB were significantly reduced by about twofolds and fivefolds compared to TX01, respectively , TX01ΔyefM-yoeB displayed higher maximum cell density than TX01, while TX01ΔyoeB exhibited growth patterns similar to that of TX01  and cultured for another 1 h, then the number of the viable bacteria was determined by plate counting. The result showed that under the condition of oxidative pressure, the survival amount of TX01ΔyoeB was comparative to that of TX01, but the survival amount of TX01ΔyefM-yoeB was significantly lower than that of TX01  or acidic LB medium (pH = 4) and cultured for another 1 h, then the number of the viable bacteria was determined. The result showed that survival rate of three strains  did not displayed significantly difference (data not shown), which indicates YefM-YoeB TA system is not related to acid tolerance.Oxidative stress and acid stress are unavoidable challenge for pathogenic bacteria invading and living in the host. To check the role of this TA system in oxidative stress, TX01, TX01Δ of TX01 . The resE. piscicida , the survival rate of TX01- and TX01ΔyoeB-infected fish was 29 and 20%, respectively, which was significantly higher than that of TX01ΔyefM-yoeB-infected fish (0%) (E. piscicida.To detect the effect of ish (0%) . These fyefM-yoeB, the complementary strain of yefM-yoeB mutant TX01ΔyefM-yoeB, TX01ΔyefM-yoeBC, was constructed. In contrast to TX01ΔyefM-yoeB, TX01ΔyefM-yoeBC exhibited a comparative resistance against oxidative stress and host serum killing and a comparative capacity of biofilm growth to those of TX01 , pTYefM-YoeB (expressing YefM-YoeB) and control plasmid pT were transformed into DH5α/pSCP345, respectively. On the same X-gal plate, the blue of DH5α/pSCP345/pTYefM was slightly weaker than that of DH5α/pSCP345/pT, and DH5α/pSCP345/pTYefM-YoeB was obviously weaker than that of DH5α/pSCP345/pTYefM  systems in pathogenic bacteria . The TA ibiotics . Compare00-folds . Studiesroteases . In thisKlebsiella pneumoniae were involved in response to oxidative stress (E. coli, upon oxidative stress, antitoxin MqsA is degraded and toxin MqsR was liberated, which enriched ghoT mRNAs as well as other stress-related transcripts and leaded to the formation of persister cells (S. pneumoniae leads to reduced resistance to oxidative stress (S. pneumoniae, we found the deficiency of the whole TA system yefM-yoeB impaired the ability of E. piscicida to resist oxidative stress, but only the inactivation of yoeB had no effect on resistance against oxidative stress, which indicates YefM perhaps plays an important role in oxidative pressure reaction, since deletion of yoeB releases YefM. In contrast to the result of oxidative stress, the deletion of whole TA system YefM-YoeB leads to enhance resistance of E. piscicida to high temperature stress and host serum stress. These observations in E. coli suggest that YefM-YoeB functions in adaptation to temperature stress (Response to environmental stress is one of prominent functions of TA systems. Under pressure, the labile antitoxins are proteolytically degraded, which releases toxins. Exposed toxins inhibit bacterial growth and help bacteria survival in adversity . For sure stress . In E. cer cells . Howeverer cells . On the e stress . Consiste stress . As far mazF toxin drives to the inhibition of biofilm formation (higA mutant and higB overexpression strain reduced the biofilm formation, which indicates HigB represses biofilm formation of P. aeruginosa (yefM-yoeB operon in S. pneumoniae bring about a considerable decrease in the thickness of the biofilm (YoeB did not affect the biofilm growth, but deletion of yefM-yoeB operon exhibited an increase of biofilm formation, which indicated the participation of YefM-YoeB TA system in the biofilm of E. piscicida. However, the role of YefM-YoeB in biofilm formation in E. piscicida are contrary to the role in S. pneumoniae (Accumulating reports have confirmed that TAs are involved in multiple important biological processes, such as biofilm formation . For exaormation . In HigBruginosa . In the ruginosa . The def biofilm . Howevereumoniae , which smvfR expression in P. aeruginosa (Salmonella promotes bacterial infection in mice (yefM-yoeB affects the resistance against adversity stress, biofilm growth, and host serum killing, which are related to the virulence (E. piscicida. As we expected, in vitro experiment demonstrated that the deficiency of yefM-yoeB significantly increased the capacity of E. piscicida to adhere and invade to host cells. Consistently, in vivo experiment showed that the deletion of yefM-yoeB significantly enhanced the ability of to disseminate in the fish tissues and elevated general virulence. Introduction of an in trans-expressed yefM-yoeB restored the virulence of TX01ΔyefM-yoeB. In contrast to our results, deletion of the yefM-yoeB locus had no effect on the virulence of Streptococcus suis (yoeB also had no effect on the virulence of E. piscicida. Moreover, TX01ΔyefM-yoeB and TX01 exhibited the similar ability of survival and replication in macrophages. Taking all the above results together, we believe that YefM-YoeB participates the virulence of E. piscicida, but its mechanism is complex and different from other pathogens. In E. piscicida, we found TX01ΔyefM-yoeB displayed obvious difference with TX01ΔyoeB in stress resistance and pathogenicity. We speculate when yoeB is deleted, another toxin protein that is functionally and structurally similar to YeoB could bind with YefM, just as the finding that YeeU is structurally similar to toxins YoeB in E. coli (TX01ΔyoeB was similar to that of TX01 but different from that of TX01ΔyefM-yoeB, which is totally missing the TA system.Increasing evidences confirm that TA systems have emerged as important elements that affect the pathogenicity of many bacteria . For exaruginosa . The TA  in mice . Since tirulence , so we ecus suis . However E. coli . TherefoS. suis, RelB1, RelB2, ParD, and ParDE negatively autoregulated the transcriptions of their respective TA operons, while RelBE2 positively autoregulated its TA operon transcription (Streptomyces lividans, YefM-YoeB complex purified from E. coli could bind with high affinity to its own promoter region (E. piscicida, we also found YefM-YoeB dramatically inhibited the activity of the promoter of yefM-yoeB. Moreover, we found YefM directly bound with its own promoter. These results indicate that YefM-YoeB functions as transcriptional regulator and negatively autoregulate the its own expression.In typical type II TA systems, the antitoxin alone or the TA complex binds to the promoter and regulates self the transcription of the TA operon . In typecription . In Strer region . SimilarE. piscicida, an important pathogen threatening worldwide aquaculture industries. Our results show that the deletion of yefM-yoeB impaired the ability of E. piscicida to oxidative resistance and reduced the persistence bacteria formation ability, but enhanced the ability of biofilm formation, serum resistance, and host infection. The inactivation of yoeB has no effects on resistance to stress and virulence. YefM-YoeB negatively autoregulated its own expression and YefM can directly bind to the promoter of yefM-yoeB. This study provides new insights into the biological activity of type II TA system YefM-YoeB in aquatic pathogenic bacteria.In conclusion, we identified type II TA system YefM-YoeB in The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the ethics committee of Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. Efforts were taken to ensure that all research animals received good care and humane treatment.DMM carried out the experiments. HJG analyzed the data and revised the manuscript. YJS participated in gene knock-out experiments. HQH participated in the infection experiments. DMS participated in the experiment design and drafted the manuscript. HYH designed the experiments, interpreted results, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although the connection between the left inferior frontal gyrus (LIFG) and the left superior temporal gyrus (LSTG) has been found to be essential for the comprehension of relative clause (RC) sentences, it remains unclear how the LIFG and the LSTG interact with each other, especially during the processing of Chinese RC sentences with different processing difficulty. This study thus conducted a 2 × 2 (modifying position × extraction position) factorial analyses to examine how these two factors influences regional brain activation. The results showed that, regardless of the modifying position, greater activation in the LIFG was consistently elicited in Chinese subject-extracted relative clauses (SRCs) with non-canonical word order than object-extracted relative clauses (ORCs) with canonical word order, implying that the LIFG subserving the ordering process primarily contributes to the processing of information with increased integration demands due to the non-canonical sequence. Moreover, the directional connection between the LIFG and the LSTG appeared to be modulated by different modifying positions. When the RC was at the subject-modifying position, the effective connectivity from the LIFG to the LSTG was dominantly activated for sentence comprehension; whereas when the RC was at the object-modifying position thus being more difficult, it might be the feedback mechanism from the LSTG back to the LIFG that took place in sentence processing. These findings reveal that brain activation in between the LIFG and the LSTG may be dynamically modulated by different processing difficulty and suggest the relative specialization but extensive collaboration involved in the LIFG and the LSTG for sentence comprehension. The SRCs and the ORCs share the same lexical content but different word order; thus, there is a well-known processing asymmetry between the SRCs and the ORCs due to the word order effect. For example, English ORCs, which induced larger storage and ordering costs due to non-canonical word order , were consistently found to be more difficult to comprehend than English SRCs with canonical word order  weifanle guiding), the relative clause (RC) did not follow canonical word order (i.e. verb + object + subject), greater processing costs were required during the storage and ordering processing to construct argument-verb relations. For this reason, increased activation in and between the LIFG and the LSTG was inferred to facilitate the comprehension of the SRCs. On the other hand, for the ORCs with canonical word order , incremental integration of all the upcoming words was sufficient for the reading; hence, less activation in between the LIFG and the LSTG was involved in the processing of the ORCs compared to the SRCs.In addition to the respective roles of the LIFG and the LSTG during the processing of sentences, enhanced effective connectivity between the LIFG and the LSTG is also reported to be closely associated with more efficient processing of sentences . The LIFBased on accumulating evidence, it appeared that complex sentence processing was probably driven by word-order analysis, supported by the LIFG , 28. HowTogether, in addition to unraveling the neural correlates underlying the processing asymmetry between Chinese SRCs and ORCs, in the present study we also aimed to investigate the information flow between the LIFG and the LSTG and attempted to answer which of the directional connections between the LIFG and the LSTG could be modulated by different processing difficulty involved in different types of RC sentences.http://www.cmuh.cmu.edu.tw, Approval No. DMR100-IRB-221). Signed written informed consent was obtained from all the participants before the experiment.Nineteen healthy, right-handed native Chinese speakers were paid to participate in this study . All participants had normal or corrected-to-normal vision and no history of neurological or psychiatric disorders. The experimental protocol for the study was approved by the Institutional Review Board (IRB) of China Medical University Hospital, Taichung, Taiwan (t1 (19) = 1.311, p = 0.205; t2 (126) = 0.762, p = 0.448).In this study, there were 64 pairs of Chinese OM-SRC and OM-ORC sentences . In each list, all 64 sentences (32 in each type) were evenly divided into four sub-lists, each of which was employed in one fMRI run. Besides, as noted in the introduction, the data from our previous study  was alsoA mixed-trial fMRI design was employed in the present study, as reported in Xu et al. . Simply,The whole experiment consisted of four runs with an equal length (6 minutes) and each run contained six blocks in a pseudorandomized order. Specifically, there were two blocks of the OM-SRC sentence reading (thus each block included four sentences), two blocks of the OM-ORC sentence reading , and two blocks of VO task as illustrated in 3.The MRI images were acquired using a 3T scanner  with a 64-channel whole-head coil located at National Cheng-Chi University, Taipei, Taiwan. The participants’ heads were immobilized with a vacuum-beam pad in the scanner. Functional, blood oxygenation level-dependent (BOLD) signals were acquired with a T2*-weighted echo planar imaging (EPI) sequence with the following parameters: slice thickness of 3.4 mm and no gap, in-plane resolution of 3.4375 × 3.4375 mm, and TR / TE / flip angle = 2000 ms / 70 ms / 77°. The field-of-view was 220 × 220 mm, and the acquisition matrix was 64 × 64. Thirty-four oblique-axial slices paralleled to the anterior commissure-posterior commissure (AC-PC) line were acquired to cover the whole brain. The first five volumes of each functional run were discarded for signal equilibrium, and a total of one hundred and eighty image volumes were acquired in each of the four runs. To obtain fine-grained localization information of the fMRI activity, a high-resolution anatomical brain image of each participant was obtained using a T1-weighted sequence . This sequence used 192 sagittal slices to cover the whole brain and resulted in an isotropic spatial resolution of 1 × 1 ×1 mmth-order B-spline interpolation [The fMRI data were processed with the Statistical Parametric Mapping package (SPM8) developed by the Wellcome Trust Center for Neuroimaging at University College London. The image data (including the target sentence and the probe question parts) with error responses were excluded from the analysis. Images of each participant were first adjusted for slice timing so that each slice’s time series was temporally aligned after correcting the time difference between the slice acquisitions in a volume. Then, the image volume series was realigned to the middle image volume of each run to correct for head movements with the 5polation , 32. A mpolation , 34. Thep < 0.05, corrected for multiple comparisons using the family-wise error (FWE) [IT)c, where αIT is the alpha level for an individual test  and c is the number of comparisons. Then, the ‘con’ or contrast images of the first-level analysis were utilized for the second-level group statistics using a one-sample T-test and the group results were visualized using the xjView toolbox  to identify selected regions of interests (ROIs). As noted in the introduction, the LIFG and the LSTG were two main ROIs in this study. However, only the LIFG was preserved from the direct contrast between the SRCs and the ORCs, we thus directly extracted the peak information of the LIFG  from the direct contrast result, but extracted the peak information of the LSTG  from the whole-brain activation. Afterwards, we used MarsBaR (http://marsbar.sourceforge.net/) in SPM8 to extract the adjusted BOLD time courses of those ROIs  with a 5-mm radius for each participant [An analytical, statistical design was constructed for each participant, using the general linear model (GLM) with the parameters consisting of the onsets of target sentence and visual orientation, convolved with a canonical hemodynamic response function and its temporal derivative , 36. Theor (FWE) . The FWEticipant .p < 0.01. Finally, the binomial test with a significance level of p < 0.05 was used to further summarize the group results regarding the directional connections between the LIFG and the LSTG [The Granger causality (GC) is based on the delayed version of the time courses for deriving the temporal dependence . This isthe LSTG . Moreovethe LSTG .t (18) = -1.874, p = 0.077). As for RT (mean ± SD), the contrast between the OM-SRC (1308.66 ± 241.77 ms) and OM-ORC (1292.2 ± 200.48 ms) conditions did not result in any significant difference (t (18) = 0.262, p = 0.797). For the SM-RC sentences, the overall comprehension accuracy (mean ± SD) across participants was 94.33 ± 3.90%, with an accuracy of 94.57 ± 3.10% for the SM-SRC sentences and an accuracy of 94.08 ± 4.65% for the SM-ORC sentences. No significant difference was found between them (t (18) = 0.389, p = 0.702). With regard to RT (mean ± SD), there was also no significant difference (t (18) = - 0.060, p = 0.953) between the SM-SRC (1137.88 ± 194.55 ms) and SM-ORC (1139.17 ± 161.62 ms) sentences.The accuracy and reaction time (RT) of the responses to the comprehension questions were recorded. Only the RTs of accurate responses and within two standard deviations (SDs) around the mean were included for further analysis. For the OM-RC sentences, the accuracy (mean ± SD) across participants was 87.91 ± 7.96%. The accuracy of the OM-SRC sentences (85.69 ± 10.12%) was numerically lower than that of the OM-ORC sentences (90.13 ± 4.20%)  = 6.870, p = 0.017, ηp2 = 0.276), with OM-RCs being slower to process overall than SM-RCs, but not a significant main effect in extraction position  = 0.059, p = 0.812, ηp2 = 0.003). Additionally, no significant interaction between modifying position and extraction position was found  = 0.064, p = 0.803, ηp2 = 0.004). As for the accuracy, the non-parametric Friedman test indicated that there was a significant difference in modifying position (χ2 (3) = 17.183 p < 0.001, with a mean rank of 3.16 for SM-SRCs, 3.03 for SM-ORCs, 1.82 for OM-SRCs and 2.00 for OM-ORCs). A Wilcoxon Signed Ranks test with Bonferroni correction was further conducted to test pairwise comparison. The results showed that the accuracy of OM-SRCs was significantly lower compared to SM-SRCs  and SM-ORCs . Besides, the accuracy of OM-ORCs was also statistically lower than that of SM-SRCs  and SM-ORCs . However, there were no significant differences between SM-SRCs and SM-ORCs  and between OM-SRCs and OM-ORCs .To see how the modifying position and extraction position affect the comprehension performance, we first normalized the behavioral data collected from the SM-RC and OM-RC experimental trials by converting the measures to z-scores. The results are depicted in Therefore, the OM-RC sentences were found to be significantly harder to comprehend than the SM-RC sentences because of the lower accuracy and also slower RT in the OM-RC sentences. Besides, regardless of the modifying position, SRCs seemed to be comparable with ORCs, although the sentences with ORCs showed some numerical advantage (relatively higher accuracy and faster RT) than the sentences with SRCs.p < 0.05 (corrected by FWE) for each type of sentences in contrast with the control VO task.As shown in We further conducted a direct contrast analysis on each pair of the SM-RC sentences as well as each pair of the OM-RC sentences to highlight the brain activation associated with sentence comprehension. As shown in p = 0.0318, binomial test) rather than the SM-ORC sentences . On the other hand, the GC from the LSTG to the LIFG was far from significance in both conditions . Moreover, the Wilcoxon Signed Ranks Test indicated that the strength of the GC from the LIFG to the LSTG in the SM-SRC condition was significantly stronger than that in the SM-ORC condition ; by contrast, the strength of the GC from the LSTG to the LIFG revealed no significant difference between both conditions . Besides, during the processing of the SM-SRC sentences, the connection from the LIFG to the LSTG was found to be significantly stronger than that in the opposite direction of these two brain areas .The GC results showed that the LIFG significantly Granger-caused the LSTG during the comprehension of the SM-SRC , whereas the GC from the LIFG to the LSTG was far from significant in both conditions . On the other hand, the Wilcoxon Signed Ranks Test showed that there was no significant difference in the connectivity strength between the OM-SRCs and OM-ORCs . However, the strength of the connectivity from the LSTG to the LIFG was significantly stronger than that of the opposite direction during the processing of the OM-SRCs  and also the OM-ORCs . Therefore, the feedback mechanism from the LSTG to the LSTG might play primary role in the comprehension of the OM-RC sentences.On the other hand, for the OM-RC sentences, in view of the important role of the LSTG in the processing of RC sentences see , we alsoF  = 32.390, p < 0.001, ηp2 = 0.643), with enhanced activation for the comprehension of the OM-RC compared to the SM-RC sentences. There was also a significant main effect in extraction position  = 4.399, p < 0.05, ηp2 = 0.196), with the sentences with SRCs inducing greater activation in the LIFG overall than the sentences with ORCs. However, no significant interaction between modifying position and extraction position was found  = 2.538, p = 0.129, ηp2 = 0.124). On the other hand, for the activation in the LSTG, there were significant main effects in modifying position  = 19.528, p < 0.001, ηp2 = 0.520) and extraction position  = 16.607, p = 0.001, ηp2 = 0.480). Specifically, greater activation in the LSTG was significantly recruited during the comprehension of the OM-RC than the SM-RC sentences. The sentences with SRCs tend to involve greater activation in the LSTG compared to the sentences with ORCs. In addition, there was a significant interaction between modifying position and extraction position  = 17.836, p = 0.001, ηp2 = 0.498). Post-hoc analysis with Bonferroni correction further showed that a significant activation difference in the LSTG was only observed when the RC was located at the subject-modifying position (p < 0.001) rather than the object-modifying position (p = 0.675).To explore how the modifying position and extraction position influence regional brain activation during sentence processing, we conducted a two-way ANOVA on the data from the SM-RC and OM-RC sentences to test the significance of the activation difference in the LIFG and the LSTG. The results showed that for the activation in the LIFG, there was a significant main effect in modifying position (χ2 (3) = 17.1337, p = 0.001, with a mean rank of 3.53 for the SM-SRCs, 2.21 for the SM-ORCs, 1.89 for the OM-SRCs and 2.37 for the OM-ORCs). A Wilcoxon Signed Ranks test with Bonferroni correction further indicated that stronger connectivity from the LIFG to the LSTG was significantly involved in the processing of the SM-SRCs in comparison to the SM-ORCs . Meanwhile, the strength of the connectivity from the LIFG to the LSTG did not result in any significant difference between the OM-SRCs and the OM-ORCs . On the other hand, the strength of the connectivity from the LSTG to the LIFG did not result in any significant differences across sentences (χ2 (3) = 4.074, p = 0.254).Regarding the information flow between the LIFG and the LSTG, the results showed that the connectivity from the LIFG to the LSTG rather than the opposite direction was significantly involved in the reading of the SM-RCs but only associated with the SM-SRCs. On the other hand, the feedback connection from the LSTG to the LIFG was engaged in the comprehension of both the OM-SRC and OM-ORC sentences see . To be nTherefore, the abovementioned brain activations, as well as the connectivity findings, might indicate different neural mechanisms involved in the processing of RC sentences with different complexity Click here for additional data file.


To investigate the clinical characteristics of patients with intraocular lens (IOL) dislocation after IOL implantation in Chinese Han populations.  The medical records of patients with IOL dislocation were retrospectively reviewed from January 2007 to December 2017, and a total of 312 patients  (328 eyes) were included in this study. The axial length (AL), IOL power, and the time interval between cataract surgery and IOL dislocation as well as the ocular conditions associated with IOL dislocation were recorded. The IOL dislocation was classified and graded based on its relationship with the capsule and the position of the dislocated IOL. 2 = 33.3992, P < 0.001); ocular blunt traumas were significantly higher in in-the-bag dislocations, while open-globe injuries were significantly higher in out-of-the-bag dislocations. A statistically significant difference was found for the ratio of patients with AL longer than 30 mm between in-the-bag dislocation and out-of-the-bag dislocation .  The mean time between original cataract surgery and IOL dislocation was 5.63 ± 5.13 years; IOL dislocation occurred in up to 56.1% (184 eyes) of the eyes within 5 years. Trauma was found in 136 eyes (41.5%); pars plana vitrectomies were performed in 61 eyes (18.6%), and high myopia was detected in 108 eyes (32.9%). A total of 243 eyes (74.1%) had out-of-the-bag IOL dislocations, while 85 eyes (25.9%) had in-the-bag IOL dislocations. There was a statistically significant difference in the constituent ratio of trauma between in-the-bag dislocation and out-of-the-bag dislocation (Pearson's chi In Chinese Han populations, the most common IOL dislocation is out-of-the-bag dislocation; the most common risk factors were trauma, long axial length, and eyes undergoing pars plana vitrectomy; a minimum follow-up of 5 years is suggested for IOL dislocation-predisposed eyes undergoing cataract surgery. Dislocation of an intraocular lens (IOL) is uncommon but is one of the most serious complications following cataract surgery . It is aThe causes of IOL dislocation include the absence of adequate capsular or zonular support. Previously reported factors associated with zonular weakness or dehiscence and capsular insufficiency were indicated in eyes with pseudoexfoliation syndrome , uveitisIn this study, a retrospective chart review of consecutive patients with IOL dislocation was performed, and the clinical characteristics of IOL dislocation patients in Chinese Han populations were demonstrated and analyzed.We retrospectively reviewed all charts of subjects with IOL dislocation after IOL implantation surgery between January 2007 and December 2017 in Shanghai, and those who required further surgical intervention were included. This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of the Eye & ENT Hospital of Fudan University.Basic information, such as age and sex, axial length (AL), intraocular pressure (IOP), corneal endothelial cell density, corneal astigmatism, time for cataract surgery, time between IOL dislocation and surgical intervention, duration of IOL dislocation, and surgical choices for IOL dislocation, was acquired and demonstrated. Associated ocular conditions when the patients presented to the clinic were also investigated.Several standards for classifying IOL dislocation have been established –15.Based on whether or not the dislocated IOL is in the capsule, it can be classified into two categories: in-the-bag dislocation and out-of-the-bag dislocation; it can also be classified as dislocation in the anterior chamber, in the posterior chamber and/or in the anterior vitreous cavity, and in the deep vitreous cavity, relying on the sites of dislocated IOL. For those whose IOL dislocated in the posterior chamber and/or the anterior vitreous cavity, the sites of dislocated IOL were further described as superior, superonasal, nasal, inferonasal, inferior, inferotemporal, and superotemporal, and the severities were graded as mild, moderate, and severe.P values less than 0.05 were considered statistically significant.SPSS software  was used for the statistical analysis. Continuous variables are presented as means ± standard deviations. Discrete variables were presented as percentages. Fisher's exact test and chi-squared test were used to analyze the discrete variables. Any differences with A total of 312 patients   were included in this study, with a mean age of 48.10 ± 20.17 years, ranging from 2 to 85 years . The inc2 = 9.7355, P value = 0.002).The mean AL was 25.65 ± 3.31 mm (17.74 to 36.69 mm). For in-the-bag dislocations, the mean AL was 26.51 ± 3.78 mm (20.86 to 35.86 mm), with 18.8% (16 eyes) of patients having an AL longer than 30 mm, 23.5% (20 eyes) between 26.0 mm and 30.0 mm, and 57.6% (49 eyes) shorter than 26 mm. For out-of-the-bag dislocations, the mean AL was 25.34 ± 3.07 mm (17.74 to 36.69 mm), with 7.0% (17 eyes) of patients having an AL longer than 30 mm, 22.6% (55 eyes) between 26.0 mm and 30.0 mm, and 70.4% (171 eyes) shorter than 26 mm . StatistAssociated ocular conditions with IOL dislocation are shown in 2 = 100.25, P value < 0.001). The ratio of open-globe injuries was significantly higher before primary IOL implantation than after primary IOL implantation, while ocular blunt trauma was significantly higher after primary IOL implantation than before primary IOL implantation.A total of 136 eyes (41.5%) were associated with trauma. Of these, 95 eyes (29.0%) were open-globe injuries, and 41 eyes (12.5%) were ocular blunt trauma. A statistically significant difference between the constituent ratio of trauma before and after primary IOL implantation was found  had ocular diseases . High myA total of 108 eyes (32.9%) had ocular surgeries conditions . Pars plA total of 42 eyes (12.8%) had a history of capsule rupture during cataract surgery; 8 eyes (2.4%) received Nd : YAG laser capsulotomy, and 21 eyes (6.4%) had capsular contraction syndrome.Detailed information on IOL dislocation classified by the aforementioned classification standard is provided in A total of 85 eyes (25.9%) were seen with in-the-bag dislocations , and 243Associated ocular conditions with in-the-bag and out-of-the-bag dislocations are shown in 2 = 33.3992, P < 0.001), and ocular blunt traumas were significantly higher in in-the-bag dislocations, while open-globe injuries were significantly higher in out-of-the-bag dislocations.The most common associated ocular conditions with in-the-bag dislocations were high myopia in 36 eyes (42.4%), trauma in 26 eyes (30.6%), and retinitis pigmentosa in 13 eyes (15.3%), while the most common associated ocular conditions with out-of-the-bag dislocations were trauma in 110 eyes (45.3%) , high myopia in 72 eyes (29.6%), and pars plana vitrectomy in 50 eyes (20.6%). There was a statistically significant difference between in-the-bag dislocation and out-of-the-bag dislocation on the constituent ratio of trauma  and traumatic vitreoretinopathy (31.1%). Vitrectomy for retinal detachment is more likely to cause IOL dislocation than vitrectomy for macular disease . Part of the reason may be that aggressive peripheral vitrectomy with scleral depression during the vitrectomy for retinal detachment may damage the posterior zonular fibers. Traumatic vitreoretinopathy is often associated with anterior or posterior capsular rupture. Also before 2011, pars plana vitrectomy used a 20-gauge vitrectomy system with a lower vitrector cutting rate (20G vitrectomy 800–2500 cuts per minute versus 23G vitrectomy 2500–7500 cuts per minute) and a larger diameter of vitrectomy probe (20G vitrectomy probe 1.0 mm versus 23G vitrectomy probe 0.5 mm) than a 23-gauge vitrectomy system, which is more likely to damage posterior zonular fibers and the posterior lens capsule. Additionally, damage to the posterior lens capsule may cause traumatic injury of the capsule integrity and, in turn, was linked to a larger percentage of out-of-the-bag dislocations in the present study. A total of 30 eyes with IOL dislocation had previously undergone surgeries for congenital cataract in this study. Because of inadequate primary posterior capsulotomy and/or anterior vitrectomy during the primary IOL implantation, the anterior vitreous face formed a scaffold for the proliferation of lens epithelial cells, which may contribute to IOL shift forward and result in IOL anterior displacement.The most common surgical techniques to treat IOL dislocation were IOL exchange and IOL refixation. The usual treatment for in-the-bag dislocations were intraocular lens complex explantation with or without new IOL sulcus fixation. However, for out-of-the-bag IOL dislocation, several factors may influence the choice of old IOL refixation or new IOL exchange: the amount of capsule support, the position and degree of the zonular dialysis, and other factors. In this study, we found that 19 eyes were subjected to two IOL repositionings before the latest sutured scleral refixation. Part of the reason is that the IOL is placed in the sulcus; however, the capsular support is inadequate, leading to repeated IOL dislocation, especially downstairs dislocation. The lens capsule may be able to temporarily hold an IOL in the sulcus but not enough for permanent fixation. We suggest that once out-of-the-bag IOL dislocation occurs, the old IOL should be removed, and a new, larger diameter optic IOL should be fixed to the sulcus. The three-piece IOL is not recommended to be used, for the rigid haptics of a three-piece IOL may aggravate further damage to the already weak lens zonule. Moreover, with the advancement of technology, numerous surgical techniques for scleral fixation of dislocated IOL have been developed, for example, an innovative scleral pockets technique for intrascleral fixation of IOL , which iIn conclusion, the most common IOL dislocation is out-of-the-bag dislocation; the most common risk factors include trauma, high myopia, and eyes undergoing pars plana vitrectomy; a minimum follow-up of 5 years is suggested for eyes predisposed for undergoing cataract surgery.